FasL (CD95L) is a type II homotrimeric transmembrane protein of the TNF (Tumor Necrosis Factor) family of cytokines (1). FasL is the ligand of the extracellular receptor designated Fas. FasL is especially expressed on activated T lymphocytes and natural killer cells, as a weapon to eliminate transformed and infected cells expressing the transmembrane receptor Fas (CD95/APO-1) (2). Binding of ligand FasL to its cellular receptor Fas triggers apoptosis via the caspase cascade. FasL itself is homotrimeric, and a productive apoptotic signal requires that FasL be oligomerized beyond the trimeric state.
In view of the interactions observed between the FasL protein and its receptor Fas, targeting human Fas initially appeared as a promising approach to treat cancer. But assays performed with an agonistic anti-Fas antibody triggered fulminant lethal hepatitis upon injection in mice, precluding the use of Fas inducers for a therapeutical purpose in human (3).
Observation that cleavage of membrane-bound FasL by a metalloprotease (4, 5) generates soluble homotrimeric FasL (sFasL), which is weakly apoptotic, and competes with membrane FasL for cell killing (6, 7) were made. Interestingly, upon cross-linking with antibodies, sFasL recovers its pro-apoptotic activity, and a FasL hexamer appears as the smallest functional form (8). Similarly, agonistic anti-Fas monoclonal antibodies (mAb) are mostly of the IgM or the self-aggregating IgG3 isotypes. In an attempt to avoid the need for cross-linking reagent, the inventors prepared chimeric molecules as polymers of FasL extra cellular domain (FasL chimeras) which proved to be non toxic and harboured cytotoxic, especially apoptotic, properties.